Dopaminergic (DA) neurotransmission from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) plays a crucial role in regulating alcohol drinking behavior. Acute exposure to ethanol has been shown to increase dopamine levels in the NAc, through direct effects on DA neuron excitability, with chronic exposure resulting in reduced levels, a mechanism believed to be the basis for drug dependence. However, few studies have explored the role of ethanol on GABAergic regulation of VTA activity. Here we present preliminary findings showing an ethanol-induced enhancement of GABA release onto VTA-DA neurons. Using a combination of electrophysiology and two-photon laser scanning microscopy, the goals of the proposed experiments are to (1) determine the mechanism by which ethanol enhances inhibitory GABAergic neurotransmission in the rat VTA and (2) determine how this ethanol-induced enhancement in GABA transmission relates to the actions of naltrexone, a CNS-acting drug currently approved to treat alcoholism, in blocking ethanol-induced increases in NAc dopamine levels. Using the visual slice patch-clamp preparation, recordings of GABA-mediated miniature inhibitory postsynaptic currents (mlPSCs) will be made from VTA-DA neurons of the rat in the presence of ethanol and under various conditions. This technique will be employed to determine the effect of altered extracellular and intracellular calcium conditions on ethanolelicited GABA release from presynaptic terminals. Additionally, two-photon laser scanning fluorescence micrsocopy will be employed to visualize changes in calcium flux in presynaptic nerve terminals synapsing onto eGFP-labeled DA neurons in the mouse VTA. Additionally, the role of the serotonin subtype receptor, 5-HT2C, will be assessed on the ethanol-induced enhancement in GABA release using mlPSC recordings. The second half the proposal is dedicated to determining the modulatory role of presynaptic mu-opiate receptors (MORs) on the ethanol-induced increase in GABA release. The aim is to demonstrate that endogenous opiates, acting on MORs to inhibit GABA release, masks the ethanol-ethanol induced increase in GABA release. This will be determined using a combination of mlPSC recordings and cell-attached current-clamp recordings of DA neuron firing rate. The findings from the proposed experiments will increase our understanding of ethanol modulation of neurotransmission in the region of the brain important for drug and alcohol dependence and may additionally increase our understanding of the therapeutic utility of the naltrexone in preventing relapse in recovering alcoholics. [unreadable] [unreadable] [unreadable]